DEVELOPMENT OF INTIMAL HYPERPLASIA IN TRANSPLANT ARTERIOSCLEROSIS

83 Pages·2003·0.99 MB·English

The Department of Surgical Sciences
Karolinska Institutet, Stockholm, Sweden

DEVELOPMENT OF
INTIMAL HYPERPLASIA IN
TRANSPLANT
ARTERIOSCLEROSIS
Piotr Religa

Stockholm 2003
All previously published papers were reproduced with permission from the publisher.

Published and printed by Repro Print AB
Stockholm, Sweden
© Piotr Religa, 2003
ISBN 91-7349-448-8
ABSTRACT
Vascular disease is the main cause of disability and mortality in the western world and the major limiting
factor for long-term survival of transplanted organs. Occlusive vascular lesions lead to ischemia and
structural changes in organs and in transplant arteriosclerosis and restenosis after endovascular procedures,
narrowing of the vessel lumen is partly due to intimal hyperplasia caused by smooth muscle cells (SMCs).
In this thesis, I have examined the mechanisms involved in intimal hyperplasia in a rat aortic allograft
model of transplant arterioscleroris with an emphasis on the biology of SMCs in this process.
First, the phenotypic properties of the SMCs involved in the formation of intimal hyperplasia were studied
by electron microscopy and immunohistochemistry. A segment of the abdominal aorta was transplanted
orthotopically from Fischer to Lewis rats and the transplanted vessels examined after 1-12 weeks. After 1
week, loss of endothelial cells, adhesion of platelets and leukocytes to the luminal surface, and a
phenotypic modification of SMCs in the media were observed. Subsequently, modified SMCs appeared in
the intima and lymphocytes and macrophages were found to infiltrate the intima as well as the adventitia.
This occurred together with detachment of endothelial cells and activation of SMCs in the media as
determined by the induction of cellular retinol-binding protein-1. Later, SMα-actin positive SMCs were
observed to migrate into the intima and proliferate as judged by staining for cyclin D1 and proliferating
cell nuclear antigen. TUNEL- and Fas/CD95-positive SMCs, indicating apoptosis, appeared in the media
which was followed by a reduction in SMα-actins taining in this layer. The continued development of the
neointima was associated with a decrease in SMα-actin-positive SMCs, an increased staining for the
extracellular matrix components fibronectin and osteopontin, and a further accumulation of inflammatory
cells. The maximum growth in size of the neointima with an increase both in the number of SMCs and the
content of extracellular matrix occurred 4-8 weeks after transplantation. SMCs and
monocytes/macrophages in the neointima and in the media were also noted to accumulate lipid, turn into
foam cells, and eventually show signs of necrosis and apoptosis. Within the lipid-rich cell remnants,
calcification also occurred. Finally, after 12 weeks, the growth in mass of the intimal lesions ceased and
reformation of an endothelial lining was detected.
In order to examine if SMCs derived from the host animal can participate in intimal hyperplasia, we
transplanted aorta of F344 female rats to Lewis male rats with or without cyclosporin A treatment. As a
control, one group of animals was transplanted with aortic isografts exposed to prolonged cold ischemia.
Infiltration of SMCs and inflammatory cells into the intimal lesions, cell proliferation, and apotosis were
analyzed by immunostaining and laser microdissection followed by real-time PCR for the SRY gene to
determine cell origin. Early after transplantation, proliferating and apoptotic graft SMCs were observed in
the neointima and leukocytes and immunoglobulins appeared in the grafts. At this time apoptosis of
medial SMCs occurred and proliferating, SRY-positive, host-derived SMCs started to accumulate in the
neointima. After 8 weeks, the neointima was mainly composed of host-derived SMCs. Immunosupression
with cyclosporin A significantly decreased the number of host SMCs in the neointima and only a small
number of host SMCs were observed in isografts exposed to prolonged ischemia. To explore the possible
bone marrow origin of allograft cells, female LEW rats were irradiated and substituted with bone marrow
from male LEW rats by transplantation of vascularized bone marrow or by infusion of bone marrow cells,
followed by transplantation of aorta from female F344 rats. Immunostaining for cell-specific markers and
real-time PCR for the SRY gene showed that the number of leukocytes was lower than the number of
bone marrow-derived cells in intimal lesions. Primed in situ labeling for the SRY gene combined with
immunostaining confirmed the presence of SM-like cells of male origin in the vessel wall in the intima.
Similar observations were made after balloon injury of the carotid artery.
The findings add further support for the prior assumption that early loss of endothelial cells contribute to
the initial response and activation of SMCs, whereas the inflammatory process may be the dominating
factor that influence vessel structure later after transplantation. The observations also suggest that
progenitors of bone marrow origin give rise to cells with SM-like properties during the formation of
intimal hyperplasia after allotransplantation as well as after balloon injury. In addition, the results provide
the basis for a novel theory of transplant arteriosclerosis which suggests that the development of intimal
hyperplasia in this vasculopathy is a dynamic two-stage process that involves apoptosis of resident graft
SMCs triggered by an allogenic immune response which also promotes recruitment of host-derived SMCs.

©Piotr Religa, 2003,
ISBN 91-7349-448-8
To my family

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LIST OF PUBLICATIONS

The thesis is based on the following original articles, referred to in the text
by their Roman numerals

I. Bojakowski, K., Religa, P., Bojakowska, M., Hedin, U., Gaciong, Z., and
Thyberg, J. 2000.
Arteriosclerosis in Rat Aortic Allografts: Early Changes in Endothelial
Integrity and Smooth Muscle Phenotype
Transplantation 70:65-72

II. Religa, P., Bojakowski, K., Gaciong, Z., Thyberg, J., and Hedin, U. 2002.
Arteriosclerosis in Rat Aortic Allografts: Dynamics of Cell Growth,
Apoptosis and Expression of Extracellular Matrix Proteins
Molecular and Cellular Biochemistry, in press.

III. Religa, P., Bojakowski, K., Maksymowicz, M., Bojakowska, M., Sirsjö, A.,
Gaciong, Z., Olszewski, W., Hedin, U., and Thyberg, J. 2002.
Smooth Muscle Progenitor Cells of Bone Marrow Origin Contribute to
the Development of Neointimal Thickenings in Rat Aortic Allografts and
Injured Rat Carotid Arteries
Transplantation 74, 1310-1315

IV. Religa, P., Bojakowski, K., Gaciong, Z., Thyberg, J., and Hedin, U. 2002.
Intimal Hyperplasia in Rat Aortic Allografts: Role of Allogenic Immune
Response in the Recruitment of Host Derived Smooth Muscle Cells
Manuscript submitted

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CONTENTS
Abstract...................................................................................................................................................1
List of publications................................................................................................................................3
Contents..................................................................................................................................................4
List of abbreviations..............................................................................................................................5
Introduction...........................................................................................................................................7
Normal arteries...............................................................................................................................9
Restenosis......................................................................................................................................10
Transplant arteriosclerosis and the mechanisms of transplant rejection............................10
Atherosclerosis.............................................................................................................................15
Comparison of transplant arteriosclerosis, atherosclerosis and restenosis.........................17
Intimal hyperplasia.......................................................................................................................17
Activation and proliferation of medial SMCs..................................................................19
Migration of SMCs...............................................................................................................21
Formation of the intimal lesion..........................................................................................21
Smooth muscle cells....................................................................................................................22
SMC progenitor cells...................................................................................................................24
Aims.......................................................................................................................................................28
Materials and methods........................................................................................................................30
Transplantation procedures of rat aorta...................................................................................30
Bone marrow transplantation.....................................................................................................30
Rat carotid balloon injury model...............................................................................................32
Immunohistochemistry...............................................................................................................32
Detection of calcium phosphate................................................................................................33
Laser capture microdissection....................................................................................................33
Real-time PCR..............................................................................................................................33
In situ tunel...................................................................................................................................34
In situ detection of chromosome Y..........................................................................................34
Confocal microscopy...................................................................................................................35
Electron microscopy....................................................................................................................35
Statistics.........................................................................................................................................36
Results...................................................................................................................................................38
General description......................................................................................................................38
Early changes in the structure of aortic allografts..................................................................38
Endothelial damage..............................................................................................................38
Activation of SMC................................................................................................................39
Formation of neointima..............................................................................................................41
Proliferation of SMCs in the neoitnima............................................................................42
Diversity of SMCs................................................................................................................42
Diverse origin of SMCs in formation of intimal lesions................................................44
Medial destruction and apoptosis..............................................................................................46
Is the accumulation of host-derived SMCs the result of an allogenic immune response?48
General discussion and conclucions.................................................................................................50
The general features of transplant arteriosclerosis.................................................................50
Development of intimal hyperplasia and the biology of SMCs............................................52
Biphasic evolution of transplant arteriosclerosis....................................................................53
Future directions..................................................................................................................................56
Acknowledgements.............................................................................................................................59
References.............................................................................................................................................61

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LIST OF ABBREVIATIONS
CMV cytomegalovirus
CRBP-1 cellular retinol-binding protein-1
FGF fibroblast growth factor
FISH fluorescent in situ hybridization
HLA human leukocyte antigen
ICAM intercellular adhesion molecule
IFN interferon
IGF insulin-like growth factor
IL interleukin
LCM laser capture microdissection
LDL low-density lipoprotein
LSCM laser scanning confocal microscopy
MCP-1 macrophage chemoattractant protein-1
MHC major histocompatibility complex
MMP matrix metalloproteinase
PCNA proliferating cell nuclear antigen
PDGF platelet-derived growth factor
PRINS primed in situ labelling
PTCA percutaneous transluminal coronary angioplasty
RT PCR real-time polymerase chain reaction
SM smooth muscle
SMC smooth muscle cell
SRY sex related gene on chromosome Y
TGF transforming growth factor
TNF tumor necrosis factor
TUNEL Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling
VCAM vascular cell adhesion molecule
vWf von Willebrand factor

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INTRODUCTION
Replacement-organ transplantation is an established treatment for end-stage disease of
the kidneys, heart, liver or lungs. With respect to kidney transplantation, which is the
most common solid-organ transplantation, the procedure gives improved quality of life
and higher survival rates for patients in comparison with dialysis. About 200 000
transplants from cadaver- and living donors have been performed every year in the USA
since the first successful kidney transplantation in 1954. The 1-year survival rate of renal
grafts is about 80-90% and the half-life is about 10 years (Cecka, 2000). The first cardiac
transplantation was done by Dr. Christian Barnard in 1967 and has since become the
treatment of choice for patients with severe cardiac dysfunction and life expectancy of
less than 6-12 months. After cardiac transplantation, the survival rate is up to 85% after
1 year and 66% after 5 years (Keck et al., 1997).
Transplanted organs evoke an immune response with both cellular and humoral
components, directed against tissue antigens incompatible with those of the recipient
(Hancock et al., 2002; Mauiyyedi and Colvin, 2002). Therefore, recognition and rejection
of the allograft by the immune system of the recipient has to be prevented by
immunosuppressive drugs. The progressive development of immunosuppressive therapy
after transplantation, for example through the introduction of azathioprine and
prednisone in the sixties, cyclosporin in the eighties, and sirolimus and mycophenolate
mofetil in the nineties has constantly improved the survival of grafts and patients, mostly
with respect to short-term results (Gonin, 2000; Olyaei et al., 2001). However, the long-
term survival after transplantation is still limited and a progressive deterioration of graft
function occurs in 3-5% of all transplanted patients per year (Cecka, 2000; Keck et al.,
1997; Wilkinson, 2001).
Chronic transplant dysfunction is the major limiting factor for successful outcome after
transplantation of solid organs (Womer et al., 2000). The process involves occlusive
arteriopathy known as transplant arteriosclerosis and atrophy with fibrosis of the organ
parenchyma (Furness, 2001; Tejani and Emmett, 2001). Transplant arteriosclerosis is, in
general terms, a reparatory remodeling of vessels in transplanted solid organs (also
referred to as transplant vasculopathy). The process is characterized by infiltration of
inflammatory cells in the vessel wall, a progressive narrowing of the vessel lumen due to
a healing reaction in the intima, intimal hyperplasia, and deterioration of the media with
cellular death and fibrosis (Furness, 2001).
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The development of transplant arteriosclerosis has many features in common with other
vascular diseases such as atherosclerosis and luminal narrowing after vascular
interventions (Newby, 2000; Pasterkamp et al., 2000). Atherosclerosis is the most
common cause of arteriosclerosis and cardiovascular diseases. Also this disease process
can be regarded as a reparatory reaction to a vascular injury and begins with the
formation of lipid streaks and inflammation in the artery wall. It then involves further
lipid deposition and thickening of the inner layer of the arteries, the tunica intima. This
gives rise to a lesion, an atherosclerotic plaque, that projects into the vessel lumen, and
eventually restricts the flow of blood, leading to end-organ ischemia, for example in the
heart (Ross, 1999). Cardiovascular diseases due to atherosclerosis are the main cause of
death and disability in economically developed countries, even though the situation has
improved since the seventies due to changes in living habits and development of new
medical treatments (Oberman et al., 1994). However, it was recently concluded that a
predicted increased survival of the general population will lead to increased morbidity
and mortality because of cardiovascular disease and generate a rise in medical costs as
well as a decrease in the quality of life (Amouyel, 2002; Mukherjee et al., 2002). The main
risk factors for atherosclerosis are age, high blood pressure, smoking, high concentration
of cholesterol and triglycerides in the blood, diabetes, obesity, and low physical activity.
The morbidity is higher among men as well as some ethnic groups. Furthermore, it can
be affected by drugs, narcotics, alcohol and hormonal replacement therapy (Sharabi et al.,
2001). There is no established curative therapy for atherosclerosis available, although
treatment of heart and extremity ischemia includes many pharmacological and surgical
approaches. Albeit these efforts do not cure the underlying atherosclerotic disease, they
have been shown to be effective in secondary prevention and in relief of the ischemic
symptoms, thereby significantly altering morbidity and mortality in cardiovascular disease
(Dawson et al., 2002). Vascular interventions, such as bypass surgery and percutaneous
transluminal coronary angioplasty (PTCA), with or without stents, are the most common
treatments of myocardial ischemia today (Mulukutla and Cohen, 2002; Yutani et al.,
1999). The number of vascular interventions by intravascular catheterisation is increasing
and has recently reached about 1 million interventions per year worldwide (Bittl, 1996).
Even if the early outcome is very promising, about 30-50% of patients after PTCA and
10-15% after bypass surgery need a renewed surgical treatment within one year because
of a recurrent narrowing due to intimal hyperplasia and vessel remodeling, also referred
to as restenosis and graft stenosis (Sallam et al., 2001).
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